Process for producing L-amino acids by fermentation

ABSTRACT

Culturing an L-amino acid producing microorganism belonging to the genus Brevibacterium or Corynebacterium and having a resistance to a peptide containing glutamic acid or aspartic acid gives L-amino acids in high yield.

This application is a Continuation of application Ser. No. 08/035,087 filed on Apr. 12 1993, (abandoned), which is a Continuation of Ser. No. 07/758,509, filed on Sep. 6, 1991, (now U.S. Pat. No. 5,294,547), which is a Continuation of Ser. No. 07/464,385, filed on Jan. 12, 1990, abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a process for producing L-amino acids by fermentation and microorganisms for producing L-amino acids.

2. Discussion of the Background

L-amino acids have been widely used as seasonings, medical drugs, feed additives, chemicals, reagents and the like. L-amino acids, which are produced by fermentation on an industrial scale, include L-glutamic acid, L-lysine, L-glutamine, L-arginine, L-phenylalanine, L-alanine, L-threonine, L-isoleucine, L-histidine, L-proline, L-valine, L-serine, L-ornithine, L-citrulline, L-tyrosine, L-tryptophan and L-leucine, etc. As microorganisms utilized for the production of L-amino acids, there are those belonging to the genus Brevibacterium, the genus Corynebacterium, the genus Bacillus, the genus Escherichia, the genus Seratia, the genus Providencia, and the genus Arthrobacter, etc.

It is important to produce L-amino acids at low costs on an industrial scale by enhancing the fermentation yield and accumulation of L-amino acids. To produce L-amino acids industrially at low costs utilizing these various microorganisms, improved breeding of microorganism has often been used. That is, the L-amino acid production of wild strains per se is extremely poor in many instances, and therefore, methods for imparting nutrient auxotrophy, imparting analog resistance or imparting nutrient auxotrophy in combination with analog resistance, through artificial mutation; or potentiating a gene for amino acid biosynthesis, etc. by genetic recombination, and the like are used to increase the L-amino acid productivity of the wild strain. However, fermentation with conventional strains does not produce L-amino acids in a sufficiently high yield.

Thus, there remains a need for a process which will produce L-amino acids by fermentation in high yield. There also remains a need for microorganisms which produce L-amino acids in high yield.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide a process for producing L-amino acids in high yield by fermentation.

It is another object of the present invention to provide microorganisms which produce L-amino acids in high yield by fermentation.

These and other objects, which will become apparent during the course of the following detailed description, have been achieved by the inventors' discovery that culturing strains having a resistance to a peptide containing glutamic acid or aspartic acid of the formula x-glu, glu-x, x-asp or asp-x, in which x represents an amino acid, produces L-amino acids in improved yields.

That is, one embodiment of the present invention is a process for producing an L-amino acid, which comprises culturing an L-amino acid-producing microorganism belonging to the genus Brevibacterium or the genus Corynebacterium and having a resistance to a peptide containing glutamic acid or aspartic acid in a liquid medium, for a sufficient time to accumulate the L-amino acid in the culture, and collecting the L-amino acid from the culture.

In another embodiment, the present invention relates to L-amino acid-producing microorganisms belonging to the genus Brevibacterium or the genus Corynebacterium and having a resistance to a peptide containing glutamic acid or aspartic acid.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The term L-amino acid as used herein includes L-glutamic acid, L-glutamine, L-lysine, L-arginine, L-phenylalanine, L-threonine, L-isoleucine, L-histidine, L-proline, L-valine, L-serine, L-ornithine, L-citrulline, L-tyrosine, L-tryptophan and L-leucine, etc. The present invention can even be applied to L-amino acids other than those exemplified herein so long as they are L-amino acids which can be produced by fermentation.

Examples of the peptide of the present invention include tyr-glu, ala-glu, trp-glu, met-glu, gly-glu, glu-gly, glu-leu, glu-his, gly-asp, ala-asp, asp-gly, etc. Strains having a resistance to at least one of these are referred to as peptide-resistant strains in the present invention.

The microorganism belonging to the genus Brevibacterium or the genus Corynebacterium which can be used in the present invention is a variant having the peptide resistance described above and capable of producing an L-amino acid.

To obtain the variants of the present invention, the peptide resistance described above may be induced in the parent strains described below; alternatively, the peptide resistance may also be induced in variants capable of producing an L-amino acid.

Wild strains which can be the parent strains of the variants of the present invention include bacteria belonging to the genus Brevibacterium or the genus Corynebacterium such as a Coryneform producing L-glutamic acid, and are exemplified by the following bacteria.

Brevibacterium flavum ATCC 14067

Brevibacterium lactofermentum ATCC 13869

Brevibacterium divaricatum ATCC 14020

Brevibacterium saccharolyticum ATCC 14066

Corynebacterium glutamicum ATCC 13032

Corynebacterium acetoacidophilum ATCC 13870

For the mutation of these parent strains to the variants of the present invention, a conventional method such as a treatment with N-methyl-N'-nitro-N"-nitrosoguanidine, etc. can be used. Isolation of the variants of the present invention from the mutation-treated bacterial solution can be effected by collecting the strains which can grow in a medium containing the peptide.

Media used for culturing such variants are any conventional media containing carbon sources, nitrogen sources, inorganic ions, substances satisfying nutrient auxotrophy and, if necessary, other organic trace nutrients including vitamins, etc. As carbon sources, there are preferably used carbohydrates such as glucose, sucrose, etc., organic acids such as acetic acid, etc. As nitrogen sources, there are preferably used ammonia water, ammonia gas, ammonium salts, etc. As inorganic ions, potassium ions, sodium ions, magnesium ions, phosphate ions, and the like are appropriately added to the media, as required. Incubation is preferably conducted under aerobic conditions. When the incubation is carried out while adjusting the pH of the medium to a range from 4 to 8, preferably 5 to 7.5, at a temperature of from 25° C. to 37° C., preferably 28° to 34° C., better results can be obtained. Thus, when the present strains are cultured for 1 to 7 days, remarkable amounts of L-amino acids are produced and accumulated in the media. Subsequently using a collecting method using an ion exchange resin, etc., yields crystals of L-amino acid.

Other features of the invention will become apparent in the course of the following descriptions of exemplary embodiments which are given for illustration of the invention and are not intended to be limiting thereof.

EXAMPLES

An example for the mutation of the parent strains to the variants of the present invention and the relationship between the peptide concentration and degree of growth of the present strains are shown below.

Method for Mutation.

Bacterial cells of Brevibacterium flavum ATCC 14067, which has been grown in a bouillon agar slant at 30° C. for 24 hours, were suspended in M/30 phosphate buffer solution at a cell density of 10⁹ / ml. To the cell suspension was added 200 μg/ml of N-methyl-N'-nitro-N"-nitrosoguanidine. The mixture was maintained at 0° C. for 20 minutes followed by centrifugation. The cells were inoculated on a medium having the composition shown in Table 1 and cultured at 31.5° C. for 2 to 10 days.

                  TABLE 1                                                          ______________________________________                                         Composition of Medium                                                          Component            Content                                                   ______________________________________                                         Glucose              1.0      g/dl                                             Urea                 0.2      g/dl                                             KH.sub.2 PO.sub.4    0.1      g/dl                                             MgSO.sub.4.7H.sub.2 O                                                                               0.1      g/dl                                             FeSO.sub.4.7H.sub.2 O                                                                               0.002    g/dl                                             MnSO.sub.4.7H.sub.2 O                                                                               0.002    g/dl                                             Biotin               100      μg/l                                          Thiamine hydrochloride                                                                              100      μg/l                                          tyr--glu             0.5      g/dl                                             Agar                 2.0      g/dl                                                                  (pH 7.0)                                                  ______________________________________                                    

From 20 tyr-glu-resistant strains grown in the agar medium, Brevibacterium flavum AJ 12418 (FERM BP-2205) was obtained and characterized as having high productivity of L-glutamine.

By procedures similar to the mutation above, strains having more improved productivity of amino acids could be obtained using various amino acid-producing strains as the original strain. Representative examples are shown in Table 2.

In addition to the improvement of bacteria capable of producing glutamine, lysine, arginine, glutamic acid, histidine, proline, isoleucine, etc. illustratively shown in Table 2, the present process is also effective for phenylalanine, threonine, valine, ornithine, tryptophan, citrulline, leucine, tyrosine, and serine.

The peptide resistance of the thus-obtained variants was compared with that of the parent strains.

Onto a liquid medium composed of 0.5 g/dl of glucose, 0.2 g/dl of urea, 0.15 g/dl of ammonium sulfate, 0.3 1 g/dl of KH₂ PO₄, 0.1 g/dl of K₂ HPO₄, 0.01 g/dl of MgSO₄ ·7H₂ O, 0.1 mg/dl of CaCl₂ ·2H₂ O, 100 μg/l of biotin, 100 μg/l of thiamine hydrochloride, 0.002 g/dl of FeSO₄ ·7H₂ O, 0.002 g/dl of MnSO₄ ·7H₂ O and the peptide in the amounts shown in Tables 3-9 and adjusted to a pH of 7.0, there were inoculated suspensions of each of the cells in sterile water, which were obtained by culturing in natural medium (1 g/dl of peptone, 1 g/dl of yeast extract and 0.5 g/dl of NaCl, pH 7.0) in slants for 24 hours. After culturing for 24 hours, the turbidity associated with the growth of bacteria was determined and the degree of growth is expressed in terms of relative growth degree (%) in Tables 3 through 9.

                  TABLE 2                                                          ______________________________________                                                                         Strain Having                                                        Peptide   Improved Yield                                 Amino Acid                                                                               Parent Strain                                                                              Resistance                                                                               Based On Glucose                               ______________________________________                                         Glutamine Brevibacterium                                                                             tyr--glu  Brevibacterium                                           flavum                flavum AJ 12418                                          ATCC 14067            (FERM BP-2205)                                           Corynebacterium                                                                            ala--glu  Corynebacterium                                          acetoacidophilum      acetoacidophilum                                         ATCC 13870            AJ 12419                                                                       (FERM BP-2206)                                 Lysine    Brevibacterium                                                                             val--glu  Brevibacterium                                           lactofermentum        lactofermentum                                           AJ 3445               AJ 12420                                                 (FERM P-1944)         (FERM BP-2207)                                           Corynebacterium                                                                            ala--glu  Corynebacterium                                          glutamicum            glutamicum                                               AJ 3399               AJ 12421                                                 (FERM P-1615)         (FERM BP-2208)                                 Arginine  Brevibacterium                                                                             tyr--glu  Brevibacterium                                           flavum                flavum                                                   AJ 3401               AJ 12422                                                 (FERM P-1642)         (FERM BP-2209)                                 Glutamic acid                                                                            Brevibacterium                                                                             tyr--glu  Brevibacterium                                           lactofermentum        lactofermentum                                           ATCC 13869            AJ 12423                                                                       (FERM BP-2210)                                           Corynebacterium                                                                            ala--glu  Corynebacterium                                          glutamicum            glutamicum                                               ATCC 13032            AJ 12424                                                                       (FERM BP-2211)                                 Histidine Brevibacterium                                                                             trp--glu  Brevibacterium                                           flavum                flavum                                                   AJ 3620               AJ 12425                                                 (FERM P-2316)         (FERM BP-2212)                                           Corynebacterium                                                                            glu--his  Corynebacterium                                          glutamicum            glutamicum                                               AJ 12092              AJ 12426                                                 (FERM P-7273)         (FERM BP-2213)                                 Proline   Brevibacterium                                                                             tyr--glu  Brevibacterium                                           flavum                flavum                                                   AJ 11512              AJ 12427                                                 (FERM P-5332)         (FERM BP-2214)                                 Isoleucine                                                                               Brevibacterium                                                                             ala--asp  Brevibacterium                                           flavum                flavum                                                   AJ 3686               AJ 12428                                                 (FERM P-2433)         (FERM BP-2215)                                 ______________________________________                                    

                  TABLE 3                                                          ______________________________________                                                  Peptide                                                               Gln        tyr--glu (%)   ala--glu (%)                                         Strain     0      0.05   0.1  0.3 0    0.05 0.1  0.3                           ______________________________________                                         Brevibacterium                                                                            100     85     45   0                                               flavum                                                                         ATCC 14067                                                                     Brevibacterium                                                                            100    100    100  72                                               flavum                                                                         AJ 12418                                                                       Corynebacterium                   100   70   30   0                            acetoacidophilum                                                               ATCC 13870                                                                     Corynebacterium                   100  100  100  65                            acetoacidophilum                                                               AJ 12419                                                                       ______________________________________                                    

                  TABLE 4                                                          ______________________________________                                                 Peptide                                                                Lys*      val--glu (%)   ala--glu (%)                                          Strain    0      0.05   0.1  0.3 0    0.05 0.1  0.3                            ______________________________________                                         Brevibacterium                                                                           100     95     80  10                                                lactofermentum                                                                 AJ 3445                                                                        Brevibacterium                                                                           100    100    100  90                                                lactofermentum                                                                 AJ 12420                                                                       Corynebacterium                  100   50   10   0                             glutamicum                                                                     AJ 3399                                                                        (FERM P-1615)                                                                  Corynebacterium                  100  100  100  100                            glutamicum                                                                     AJ 2421                                                                        ______________________________________                                          *When Corynebacterium glutamicum was used, 15 mg/dl of methionine was          supplemented.                                                            

                  TABLE 5                                                          ______________________________________                                                      Peptide                                                           Arg*           tyr--glu (%)                                                    Strain         0      0.05       0.1  0.3                                      ______________________________________                                         Brevibacterium flavum                                                                         100    100         30   0                                       AJ 3401                                                                        Brevibacterium flavum                                                                         100    100        100  95                                       AJ 12422                                                                       ______________________________________                                          *Liquid medium was supplemented with 5 mg/dl of guanine.                 

                  TABLE 6                                                          ______________________________________                                                  Peptide                                                               Glu        tyr--glu (%)   ala--glu (%)                                         Strain     0      0.05   0.1  0.3 0    0.05 0.1  0.3                           ______________________________________                                         Brevibacterium                                                                            100     78    142   0                                               lactofermentum                                                                 ATCC 13869                                                                     Brevibacterium                                                                            100    100     95  80                                               lactofermentum                                                                 AJ 12423                                                                       Corynebacterium                   100   75   40   0                            glutamicum                                                                     ATCC 13032                                                                     Corynebacterium                   100  100  100  94                            glutamicum                                                                     AJ 12424                                                                       ______________________________________                                    

                  TABLE 7                                                          ______________________________________                                                 Peptide                                                                His       trp--glu (%)    glu--his (%)                                         Strain    0      0.05   0.1  0.3  0    0.05 0.1  0.3                           ______________________________________                                         Brevibacterium                                                                           100    100     75   36                                               flavum                                                                         AJ 3620                                                                        Brevibacterium                                                                           100    100    100  100                                               flavum                                                                         AJ 12425                                                                       Corynebacterium                   100  100   96  55                            glutamicum                                                                     AJ 12092                                                                       Corynebacterium                   100  100  100  98                            glutamicum                                                                     AJ 12426                                                                       ______________________________________                                    

                  TABLE 8                                                          ______________________________________                                                      Peptide                                                           Pro*           tyr--glu (%)                                                    Strain         0      0.05       0.1  0.3                                      ______________________________________                                         Brevibacterium flavum                                                                         100     90        30    0                                       AJ 11512                                                                       Brevibacterium flavum                                                                         100    100        95   60                                       AJ 12427                                                                       ______________________________________                                          *Liquid medium was supplemented with 15 mg/dl of isoleucine.             

                  TABLE 9                                                          ______________________________________                                                      Peptide                                                           Ile            ala--asp (%)                                                    Strain         0      0.05       0.1  0.3                                      ______________________________________                                         Brevibacterium flavum                                                                         100     70        20    0                                       AJ 3686                                                                        Brevibacterium flavum                                                                         100    100        98   70                                       AJ 12428                                                                       ______________________________________                                    

Example 1.

An aqueous solution medium having a composition of 10% of glucose, 1% of ammonium sulfate, 0.25% of potassium primary phosphate (KH₂ PO₄), 0.04% of magnesium sulfate, 0.001% of ferrous sulfate, 350 μg/l of thiamine hydrochloride, 5 μg/l of biotin and 0.5 ml/dl of Aji-Eki® which is a soybean protein hydrolysate which accelerates the growth of L-amino acid-producing microorganisms and shortens the required culture time, at pH 7.0, was charged in an amount of 300 ml into separate small sized glass jar fermenters. After sterilizing in a conventional manner, the various L-glutamine-producing bacterial strains shown in Table 10, which had been previously grown in bouillon slants at 30° C. for 24 hours, were inoculated thereon. Then, incubation was carried out at 31.5° C. for 30 hours at 1200 rpm at an aeration rate of 1/4 volume per minute, while keeping the pH at 6.5 by the addition of ammonia gas. After completion of the fermentation, the yield of L-glutamine produced and accumulated in the solution based on glucose was determined and is shown in Table 10.

                  TABLE 10                                                         ______________________________________                                                                    Yield of                                                                       L-Glutamine                                                                    Based on                                            Strain         Property    Glucose (%)                                         ______________________________________                                         Brevibacterium Wild        29.0                                                flavum AJ 14067                                                                Brevibacterium Imparted with                                                                              40.0                                                flavum AJ 12418                                                                               tyr--glu                                                                       resistance                                                      Corynebacterium                                                                               Wild        22.5                                                acetoacidophilum                                                               ATCC 13870                                                                     Corynebacterium                                                                               Imparted with                                                                              34.0                                                acetoacidophilum                                                                              ala--glu                                                        AJ 12419       resistance                                                      ______________________________________                                    

The cells were removed from 1 liter of the solution obtained after completion of the fermentation using Brevibacterium flavum AJ 12418 by centrifugation to give a supernatant. From the supernatant, L-glutamine was isolated in a conventional manner using an ion exchange resin to give 19.0 g of L-glutimine as crystals.

Example 2.

An aqueous solution medium having a composition of 10% of glucose, 2% of ammonium sulfate, 0.1% of potassium primary phosphate, 0.04% of magnesium sulfate, 0.001% of ferrous sulfate, 200 μg/l of thiamine hydrochloride, 500 μg/l of biotin, 5 ml/dl of Aji-Eki®, 1 mg/dl of nicotinamide and 0.1% of DL-alanine, at pH 7.0, was charged in an amount of 300 ml into separate small sized glass jar fermenters. After sterilizing in a conventional manner, the various L-lysine-producing bacterial strains shown in Table 11, which had been previously grown in bouillon slants at 30° C. for 48 hours, were inoculated thereon. Then, incubation was carried out at 31.5° C. for 48 hours at 1200 rpm at an aeration rate of 1/2 volume per minute, while keeping the pH at 7.0 by the addition of ammonia gas. After completion of the fermentation, the yield of L-lysine produced and accumulated in the solution based on glucose was determined and is shown in Table 11.

                  TABLE 11                                                         ______________________________________                                                                    Yield of                                                                       L-Lysine                                                                       Based on                                            Strain        Property*    Glucose (%)                                         ______________________________________                                         Brevibacterium                                                                               AEC resistance                                                                              16.0                                                lactofermentum                                                                 AJ 3445                                                                        Brevibacterium                                                                               Imparted with                                                                               31.0                                                lactofermentum                                                                               val--glu resistance                                              AJ 12420                                                                       Corynebacterium                                                                              met, AEC resistance                                                                         23.0                                                glutamicum                                                                     AJ 3399                                                                        Corynebacterium                                                                              Imparted with                                                                               32.0                                                glutamicum    ala--glu resistance                                              AJ 12421                                                                       ______________________________________                                          *AEC = S(aminoethyl)-cystein.                                            

The cells were removed from 1 liter of the solution obtained after completion of the fermentation using Brevibacterium lactofermentum AJ 12420 by centrifugation to give a supernatant. From the supernatant, L-lysine was isolated in a conventional manner using an ion exchange resin to give 19.2 g of L-lysine as crystals.

Example 3.

An aqueous solution medium having a composition of 10% of glucose, 4% of ammonium sulfate, 0.1% of potassium primary phosphate, 0.04% of magnesium sulfate, 0.001% of ferrous sulfate, 0.001% of manganese sulfate, 100 μg/l of thiamine hydrochloride, 100 μg/l of biotin, 5 ml/dl of Aji-Eki® and 0.2% of yeast extract, at pH 7.0, was charged in an amount of 300 ml into separate small sized glass jar fermenters. After sterilizing in a conventional manner, the various L-arginine-producing bacterial strains shown in Table 12, which had been previously grown in bouillon slants at 30° C. for 24 hours, were inoculated thereon. Then, incubation was carried out at 31.5° C. for 48 hours at 1200 rpm at an aeration rate of 1/2 volume per minute, while keeping the pH at 7.0 by the addition of ammonia gas. After completion of the fermentation, the yield of L-arginine produced and accumulated in the solution based on glucose was determined and is shown in Table 12.

                  TABLE 12                                                         ______________________________________                                                                    Yield of                                                                       L-Arginine                                                                     Based on                                            Strain         Property    Glucose (%)                                         ______________________________________                                         Brevibacterium gua.sup.-, 2-thiazolyl-                                                                    25.5                                                flavum AJ 3401 alanine resistance                                              (FERM P-1642)                                                                  Brevibacterium Imparted with                                                                              32.0                                                flavum AJ 12422                                                                               tyr--glu                                                        (FERM BP-2209) resistance                                                      ______________________________________                                    

The cells were removed from 1 liter of the solution obtained after completion of the fermentation using Brevibacterium flavum AJ 12422 by centrifugation to give a supernatant. From the supernatant, L-arginine was isolated in a conventional manner using an ion exchange resin to give 17.3 g of L-arginine as crystals.

Example 4.

An aqueous solution medium having a composition of 10% of glucose, 1% of ammonium sulfate, 0.2% of potassium primary phosphate, 0.1% of magnesium sulfate, 0.001% of ferrous sulfate, 0.001% of manganese sulfate, 500 μg/l of thiamine hydrochloride, 5 μg/l of biotin and 1 ml/dl of Aji-Eki®, at pH 7.2, was charged in an amount of 300 ml into separate small sized glass jar fermenters. After sterilizing in an autoclave, the various L-glutamic acid-producing bacterial strains shown in Table 13, which had been previously grown in bouillon slants at 30° C. for 24 hours, were inoculated thereon. Then, incubation was carried out at 31.5° C. for 48 hours at 1200 rpm at an aeration rate of 1/2 volume per minute, while keeping the pH at 7.2 by the addition of ammonia gas. After completion of the fermentation, the yield of L-glutamic acid produced and accumulated in the solution based on glucose was determined and is shown in Table 13.

                  TABLE 13                                                         ______________________________________                                                                  Yield of                                                                       L-glutamic Acid                                                                Based on                                              Strain       Property    Glucose (%)                                           ______________________________________                                         Brevibacterium                                                                              Wild        44.2                                                  lactofermentum                                                                 ATCC 13869                                                                     Brevibacterium                                                                              Imparted with                                                                              49.5                                                  lactofermentum                                                                              tyr--glu                                                          AJ 12423     resistance                                                        Corynebacterium                                                                             Wild        40.1                                                  glutamicum                                                                     ATCC 13032                                                                     Corynebacterium                                                                             Imparted with                                                                              46.8                                                  glutamicum   ala--glu                                                          AJ 12424     resistance                                                        ______________________________________                                    

The cells were removed from 1 liter of the solution obtained after completion of the fermentation using Brevibacterium lactofermentum AJ 12423 by centrifugation to give a supernatant. From the supernatant, L-glutamic acid was isolated in a conventional manner using an ion exchange resin to give 35.5 g of L-glutamic acid as crystals.

Example 5.

An aqueous solution medium having a composition of 10% of glucose, 0.5% of ammonium sulfate, 0.15% of potassium primary phosphate, 0.1% of magnesium sulfate, 0.001% of ferrous sulfate, 0.001% of manganese sulfate, 300 μg/l of thiamine hydrochloride, 350 μg/l of biotin, 5 ml/dl of Aji-Eki® and 0.5% of ammonium acetate, at pH 7.0, was charged in an amount of 300 ml into separate small sized glass jar fermenters. After sterilizing in an autoclave, the various L-histidine-producing bacterial strains shown in Table 14, which had been previously grown in bouillon slants at 30° C. for 24 hours, were inoculated thereon. Then, incubation was carried out at 31.5° C. for 48 hours at 1200 rpm at an aeration rate of 1/2 volume per minute, while keeping the pH at 6.5 by the addition of ammonia gas. After completion of the fermentation, the yield of L-histidine produced and accumulated in the solution based on glucose was determined and is shown in Table 14.

                  TABLE 14                                                         ______________________________________                                                                    Yield of                                                                       L-Histidine                                                                    Based on                                            Strain       Property      Glucose (%)                                         ______________________________________                                         Brevibacterium                                                                              2-AT, sulfadiazine,                                                                          7.2                                                 flavum AJ 3620                                                                              cobalamine resistance                                             Brevibacterium                                                                              Imparted with 10.0                                                flavum AJ 12425                                                                             trp--glu resistance                                               Corynebacterium                                                                             2-AT resistance                                                                              5.0                                                 glutamicum                                                                     AJ 12092                                                                       Corynebacterium                                                                             Imparted with 9.3                                                 glutamicum   glu--his resistance                                               AJ 12426                                                                       ______________________________________                                    

The cells were removed from 1 liter of the solution obtained after completion of the fermentation using Corynebacterium glutamicum AJ 12426 by centrifugation to give a supernatant. From the supernatant, L-histidine was isolated in a conventional manner using an ion exchange resin to give 4.7 g of L-histidine as crystals.

Example 6.

An aqueous solution medium having a composition of 10% of glucose, 4% of ammonium sulfate, 0.1% of potassium primary phosphate, 0.5% of magnesium sulfate, 0.001% of ferrous sulfate, 0.001% of manganese sulfate, 100 μg/l of thiamine hydrochloride, 350 μg/l of biotin, 1 ml/dl of Aji-Eki® and 35 mg/dl of L-isoleucine, at pH 7.0, was charged in an amount of 300 ml into separate small sized glass jar fermenters. After sterilizing in a conventional manner, the various L-proline-producing bacterial strains shown in Table 15, which had been previously grown in bouillon slants at 30° C. for 24 hours, were inoculated thereon. Then, incubation was carried out at 31.5° C. for 48 hours at 1200 rpm at an aeration rate of 1/2 volume per minute, while keeping the pH at 7.0 by the addition of ammonia gas. After completion of the fermentation, the yield of L-proline produced and accumulated in the solution based on glucose was determined and is shown in Table 15.

                  TABLE 15                                                         ______________________________________                                                                    Yield of                                                                       L-Proline                                                                      Based on                                            Strain        Property     Glucose (%)                                         ______________________________________                                         Brevibacterium                                                                               ile.sup.-, sulfaguanidine                                                                   21.0                                                flavum AJ 11512                                                                              resistance                                                       Brevibacterium                                                                               Imparted with                                                                               29.0                                                flavum AJ 12427                                                                              tyr--glu resistance                                              ______________________________________                                    

The cells were removed from 1 liter of the solution obtained after completion of the fermentation using Brevibacterium flavum AJ 12427 by centrifugation to give a supernatant. From the supernatant, L-proline was isolated in a conventional manner using an ion exchange resin to give 17.5 g of L-proline as crystals.

Example 7.

An aqueous solution medium having a composition of 10% of glucose, 1% of ammonium sulfate, 0.1% of potassium primary phosphate, 0.04% of magnesium sulfate, 0.001% of ferrous sulfate, 0.001% of manganese sulfate, 100 μg/l of thiamine hydrochloride, 100 μg/l of biotin and 2 ml/dl of Aji-Eki®, at pH 7.0, was charged in an amount of 300 ml into separate small sized glass jar fermenters. After sterilizing in a conventional manner, the various L-isoleucine-producing bacterial strains shown in Table 16, which had been previously grown in bouillon slants at 30° C. for 24 hours, were inoculated thereon. Then, incubation was carried out at 31.5° C. for 48 hours at 1200 rpm at an aeration rate of 1/2 volume per minute, while keeping the pH at 7.3 by the addition of ammonia gas. After completion of the fermentation, the yield of L-isoleucine produced and accumulated in the solution based on glucose was determined and is shown in Table 16.

                  TABLE 16                                                         ______________________________________                                                                    Yield of                                                                       L-Isoleucine                                                                   Based on                                            Strain         Property*   Glucose (%)                                         ______________________________________                                         Brevibacterium AHV resistance                                                                              8.5                                                flavum AJ 3686                                                                 Brevibacterium Imparted with                                                                              13.0                                                flavum AJ 12428                                                                               ala--asp                                                                       resistance                                                      ______________________________________                                          *AHV = amino-hydroxy-valeric acid.                                       

The cells were removed from 1 liter of the solution obtained after completion of the fermentation using Brevibacterium flavum AJ 12428 by centrifugation to give a supernatant. From the supernatant, L-isoleucine was isolated in a conventional manner using an ion exchange resin to give 6.5 g of L-isoleucine as crystals.

Thus, as shown by the results given above, according to the present invention, various L-amino acids can be obtained in a good yield.

Obviously, numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that, within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein. 

What is claimed as new and desired to be secured by Letters Patent of the United States is:
 1. A process for producing an L-amino acid, which comprises culturing a mutant L-amino acid producing microorganism belonging to a genus selected from Brevibacterium and Corynebacterium which is resistant to a dipeptide at a concentration of 0.3% in a liquid medium for a time sufficient to produce said L-amino acid; andrecovering said L-amino acid produced wherein1) L-glutamine is produced from a tyr-glu resistant mutant of Brevibacterium flavum ATCC 14067 or an ala-glu resistant mutant of Corynebacterium acetoacidophilum ATCC 13870; 2) L-lysine is produced from a val-glu resistant mutant of Brevibacterium lactofermentum ATCC 13869 or an ala-glu resistant mutant of Corynebacterium glutamicum ATCC 13032; 3) L-arginine is produced from a tyr-glu resistant mutant of Brevibacterium flavum ATCC 14067; 4) L-glutamic acid is produced from a tyr-glu resistant mutant of Brevibacterium lactofermentum ATCC 13869 or an ala-glu resistant mutant of Corynebacterium glutamicum ATCC 13032; 5) L-histidine is produced from a trp-glu resistant mutant of Brevibacterium flavum ATCC 14067 or a glu-his resistant mutant of Corynebacterium glutamicum ATCC 13032; 6) L-proline is produced by a tyr-glu resistant mutant of Brevibacterium flavum ATCC 14067; and 7) L-isoleucine is produced from an ala-asp resistant mutant of Brevibacterium flavum ATCC 14067, and wherein said mutant microorganism is obtained by mutation of a parent strain, and said microorganism produces the L-amino acid in an amount greater than the amount produced by the parent strain.
 2. The process of claim 1, wherein said L-amino acid producing microorganism is obtained by contacting the parent strain with N-methyl-N'-nitro-N-nitrosoguanidine.
 3. A process for producing an L-amino acid, which comprises culturing a mutant L-amino acid producing microorganism belonging to the genus Brevibacterium which is resistant to a dipeptide at a concentration of 0.3% in a liquid medium for a time sufficient to produce said L-amino acid; andrecovering said L-amino acid produced, wherein the amino acid is L-glutamine, the mutant microorganism is a tyr-glu resistant mutant of Brevibacterium flavum ATCC 14067, the mutant microorganism is obtained by mutation of a parent strain, and the mutant microorganism produces the L-amino acid in an amount greater than the amount produced by the parent strain.
 4. The process of claim 3, wherein said L-amino acid producing microorganism is obtained by contacting the parent strain with N-methyl-N'-nitro-N-nitrosoguanidine. 